Abstract

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size (IS) in canine myocardium either given before an ischemic insult or at reperfusion similar to that produced in ischemic preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. We tested the hypothesis that EETs may play a role in these two phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 min of reperfusion before 60 min of occlusion and 3 h of reperfusion or by postconditioning with three 30-s periods of reperfusion interspersed with three 30-s periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) or its derivative, 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG), were administered 10 min before IPC, 5 min after IPC, or 5 min before POC. In a separate series, the selective EET synthesis inhibitor N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) was administered 10 min before IPC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in IS expressed as a percentage of the area at risk (AAR) [Control 21.2 ± 1.2%, IPC 8.3 ± 2.2%, POC 10.1 ± 1.8% (P < 0.001)]. 14,15-EEZE, 14,15-EEZE-PEG, and MS-PPOH markedly attenuated the cardioprotective effects of IPC and POC (14,15-EEZE and 14,15-EEZE-PEG) at doses that had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC.

cytochrome P-450 epoxygenases

epoxyeicosatrienoic acid antagonists

ischemia-reperfusion

both ischemic preconditioning (IPC) and postconditioning (POC) are fascinating endogenous mechanisms that when activated produce a powerful and, in some instances, prolonged cardioprotective effect to markedly reduce infarct size (IS) in a variety of animal species including humans (9, 18, 20). Whereas IPC is required as a pretreatment modality, which renders it less useful as a cardioprotective therapy since it is difficult to predict ahead of time when an ischemic event will occur, POC can be used once the ischemic event has started and just before and at reperfusion so its clinical potential is much greater than that of IPC (18). Despite differences in the timing of these two phenomena, there appear to be a number of similarities in the beneficial signaling pathways involved in mediating their cardioprotective mechanisms (9). These include the involvement of several receptor systems including opioid (8), adenosine (2), and bradykinin (14) receptors as well as signaling molecules and kinases such as nitric oxide (NO) (18), extracellular signal-regulated kinase (ERK) (9), protein kinase C (PKC) (13), phosphoinositide 3-kinase (PI3K)/Akt (3, 9), glycogen synthetase kinase (GSK)3β (8), ATP-sensitive potassium (KATP) channels (11, 13), and the mitochondrial permeability transition pore (MPTP) (1).

Recently, we have reported data (12) to suggest that certain arachidonic acid metabolites of the CYP epoxygenase pathway, the epoxyeicosatrienoic acids (11,12-EET and 14,15-EET), produce reductions in IS similar in magnitude to those produced by IPC when administered before ischemia or similar to POC when administered at reperfusion in canine hearts. These fascinating similarities in the timing and magnitude of protection produced by the EETs and IPC and POC have led us to hypothesize that the EETs may be an integral part of these two phenomena. Having several selective EET antagonists available (4, 5, 7) allowed us to use a pharmacological approach to address the potential role of EETs in mediating IPC and POC in the canine heart.

MATERIALS AND METHODS

This study was approved by and performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.

Materials.

14,15-EET, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), and 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG) were synthesized in the laboratory of J. R. Falck. All other chemicals were of the highest analytic or purity grades. Distilled, deionized water was used in all experiments.

General preparation of dogs.

The protocol used has been thoroughly described in detail in previous publications from our laboratory (12). Briefly, adult mongrel dogs of either sex, weighing 15–25 kg, were fasted overnight, anesthetized with the combination of barbital sodium (200 mg/kg) and pentobarbital sodium (15 mg/kg), and ventilated with room air supplemented with 100% oxygen. Body temperature was carefully controlled at 38 ± 1°C with a heating pad. Atelectasis was prevented by maintaining an end-expiratory pressure of 5–7 cm with a trap. Arterial blood pH, Pco2, and Po2 were monitored at selected intervals by an AVL blood gas analyzer and maintained within normal physiological limits (pH = 7.35–7.45, Pco2 = 30–40 Torr, and Po2 = 85–130 Torr) by adjusting the respiratory rate and the oxygen flow rate or by adding 1.5% sodium bicarbonate intravenously if necessary. An electromagnetic flowmeter (Statham 2202) was used to measure left anterior descending coronary artery (LAD) blood flow. A mechanical occluder was placed immediately distal to the flow probe, such that there were no branches between the flow probe and occluder. A double-tipped Millar (model 770) catheter was placed into the carotid artery and left ventricle (LV) to measure aortic and LV pressures and to determine LV change in pressure over time (positive and negative dP/dt). The left atrium was cannulated via the appendage for radioactive microsphere injections.

Experimental protocol.

Dogs were sequentially assigned to 14 groups for different treatments (Fig. 1). In all groups, dogs were subjected to 60 min of LAD occlusion and 3 h of reperfusion. All groups included eight animals for proper statistical analysis. In the first two groups, vehicle or 14,15-EET (0.128 mg/kg) was administered by a 2- to 3-min intracoronary infusion, as shown in the protocol (Fig. 1). In the third group, the dogs were preconditioned (IPC) by a 5-min LAD occlusion followed by 10 min of reperfusion before the 60-min prolonged occlusion period. In two subsequent IPC-treated groups, we administered one of the two EET antagonists by intracoronary injection: 14,15-EEZE (0.128 mg/kg) or 14,15-EEZE-PEG (0.128 mg/kg) 10 min before IPC or 14,15-EET. In one additional set of experiments, the EET synthesis inhibitor MS-PPOH (1.296 mg/kg) was administered 10 min before IPC only. In the POC experiments we subjected the hearts to 30 s of reperfusion interspersed with 30 s of occlusion three times before allowing the normal 3 h of reperfusion. In the two groups treated with the EET antagonists, these agents were administered into the left atrium 5 min before the first reperfusion period. In all groups, hemodynamic measurements, blood gas analyses, and regional myocardial blood flow (radioactive microspheres) measurements were performed at baseline and at 30 min into the 60-min occlusion period. After reperfusion, hemodynamics and blood gases were measured every hour, and regional myocardial blood flow was determined at the end of 3 h of reperfusion.

Infarct size determination.

IS was determined as previously described by our laboratory (7). Briefly, at the end of the 3-h reperfusion period the LAD was reoccluded and cannulated distal to the occlusion site. To determine the anatomic area at risk (AAR) and the nonischemic area, 5 ml of Patent blue dye and 5 ml of saline were injected at equal pressure into the LAD and left atrium, respectively. The heart was then immediately fibrillated and removed. The LV was dissected and sliced into serial transverse sections 6–7 mm in width. The nonstained ischemic area and the blue-stained normal area were separated and incubated in a solution containing 1% 2,3,5-triphenyltetrazolium chloride (Sigma) in 0.1 mol/l phosphate buffer, pH 7.4, at 37°C for 15 min. After incubation overnight in 10% formaldehyde, the noninfarcted and infarcted tissues within the AAR were separated and determined gravimetrically. IS was expressed as a percentage of the AAR (IS/AAR).

Regional myocardial blood flow.

Regional myocardial blood flow was measured by the radioactive microsphere technique (7). Microspheres were administered 30 min into the prolonged 60-min occlusion period and at the end of reperfusion. Carbonized plastic microspheres (15-μm diameter, New England Nuclear) labeled with 141Ce or 95Nb were suspended in isotonic saline with 0.01% Tween 80 added to prevent aggregation. The microspheres were sonicated for 5 min and vortexed for another 5 min before injection. One milliliter of the microsphere suspension (2–4 × 106 spheres) was given through the left atrial catheter and flushed by 5 ml of saline. A reference blood flow sample was drawn from the right femoral artery at a constant rate of 9.4 ml/min starting 30 s before microsphere injection and continuing for 3 min. The next day, the tissue slices were sectioned into subepicardium, midmyocardium, and subendocardium of nonischemic (3 pieces) and ischemic (5 pieces) regions. Transmural pieces were obtained from the center of several transverse sections used to determine the AAR and were at least 1 cm from the perfusion boundaries as indicated by Patent blue dye. All samples were counted in a gamma counter (Tracor Analytic 1195) to determine the activity of each isotope in each sample. The activity of each isotope was also determined in the reference blood flow samples. Myocardial blood flow was calculated by use of a preprogrammed computer to obtain the true activity of each isotope in individual samples, and tissue blood flow was calculated from the equation Qm = Qr × Cm/Cr, where Qm is myocardial blood flow (in ml·min−1·g tissue−1), Qr is the rate of withdrawal of the reference blood flow (9.4 ml/min), Cr is the activity of the blood flow sample (counts/min), and Cm is the activity of the tissue sample (counts·min−1·g−1). Transmural blood flow was calculated as the weighted average of the three layers in each region.

Exclusion criteria.

Dogs were excluded if 1) heartworms were found after the dogs were euthanized; 2) transmural collateral blood flow was >0.20 ml·min−1·g−1, 3) heart rate was >180 beats/min at the beginning of the experiment, or 4) more than three consecutive attempts were needed to convert ventricular fibrillation with low-energy DC pulses applied directly to the heart.

Statistical analysis.

All values are expressed as means ± SE. Differences between groups in hemodynamics and blood gases were compared by use of a two-way (for time and treatment) ANOVA with repeated measures and Fisher's least significant difference test if significant F-ratios were obtained. Differences between groups in tissue blood flows, AAR, and IS were compared by one-way ANOVA, and comparisons between groups were made with Fisher's least significant difference test. Differences between groups were considered significant if the probability value was P < 0.05. Linear regression analysis was performed to determine the correlation between transmural blood flow in the ischemic area and myocardial IS (IS/AAR). Analysis of covariance, with collateral flow as the covariate, was used to determine whether differences in this relationship were observed among five treatment groups selected.

RESULTS

Hemodynamics.

Mean arterial blood pressure and heart rate at baseline and at 30 min of ischemia or at the end of 3 h of reperfusion were not different among all the groups studied (Table 1). These data suggest that changes in IS were not the result of changes in myocardial oxygen demand resulting from IPC or POC or by the two EET antagonists and EET synthesis inhibitor used. We also measured pH, Po2, and Pco2 and found that these values were not different among groups at any of the times studied (data not shown).

Regional myocardial blood flow.

Transmural myocardial blood flow in the nonischemic left circumflex perfusion area and the ischemic LAD bed were measured at 30 min of occlusion and at 3 h of reperfusion with radioactive microspheres. There were no differences in nonischemic transmural blood flow among groups or transmural collateral blood flow in the ischemic bed at the measured points (Table 2). Most importantly, there were no differences in the ischemic reperfused area (area at risk, AAR) during coronary occlusion, which suggested that all groups were subjected to similar degrees of ischemia (Table 3). There were also no differences in AAR or AAR/LV among groups (Table 3). Since there were no differences in AAR, coronary collateral blood flow (Table 2), and hemodynamics (Table 1), the three major determinants of IS/AAR, it appears that IPC and POC were exerting their cardioprotective effects through other mechanisms in which the EETs may play a supporting role. Finally, in Fig. 2 we demonstrate the relationship between IS/AAR and transmural coronary collateral blood flow measured at 30 min into the ischemic period. In five groups analyzed, there was a significant (P < 0.01) inverse relationship between these two parameters, as shown by linear regression analysis. In the IPC and POC groups there was a marked parallel shift downward compared with the control group, which clearly indicates that at any given collateral blood flow one would predict a smaller IS/AAR in the IPC and POC groups. Interestingly, pretreatment with 14,15-EEZE shifted these two lines nearly back to the control group. These data further indicate that the changes observed in IS/AAR are occurring independent of changes in transmural coronary collateral blood flow.

Effects of IPC and POC on IS/AAR in absence and presence of two selective EET antagonists and EET synthesis inhibitor.

In an initial series of experiments, we decided to compare the effects of one of the major endogenous EETs, 14,15-EET, with those of IPC to determine whether the magnitude of IS reduction was similar to that which would be expected if they work via a common mechanism. The results are shown in Fig. 3A and indicate that both IPC and 14,15-EET produced nearly identical reductions in IS/AAR (IPC = 8.3 ± 2.2%, 14,15-EET = 9.0 ± 1.9%) compared with the control group (21.2 ± 0.9%). The effect of 14,15-EET was also blocked by the selective EET antagonists 14,15-EEZE (22.1 ± 1.8%) and 14,15-EEZE-PEG (21.8 ± 0.7%) as we previously demonstrated in the canine heart (7).

Effect of 14,15-EET (0.128 mg/kg) and IPC alone or when pretreated for 10 min with either of two EET receptor antagonists, 14,15-EEZE or 14,15-EEZE-PEG (0.128 mg/kg), on myocardial IS expressed as % of AAR (IS/AAR) in dogs subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. The two EET antagonists had no effect on IS/AAR by themselves but completely blocked the effect of IPC. 14,15-EET reduced IS/AAR by an amount nearly equivalent to that of IPC, and we have previously shown that 14,15-EEZE blocks the effect of 14,15-EET (7). IPC was produced by a single 5-min period of ischemia 10 min before the index ischemic period.

In Fig. 3B, we demonstrate the effect of the two selective EET antagonists 14,15-EEZE and 14,15-EEZE-PEG on the cardioprotective effect of IPC. Both 14,15-EEZE and 14,15-EEZE-PEG had no effect on IS/AAR when administered alone (14,15-EEZE = 19.8 ± 2.6%, 14,15-EEZE-PEG = 18.6 ± 1.6%) compared with the control group (21.2 ± 0.9%); however, pretreatment with both antagonists completely blocked the effect of IPC (14,15-EEZE = 20.5 ± 1.7%, 14,15-EEZE-PEG = 20.2 ± 1.8%). In addition, the EET synthesis inhibitor MS-PPOH (18.6 ± 1.4%) also abrogated the effect of IPC. These results suggest that endogenous EETs may be an important unrecognized mediator of IPC since IPC was blocked by both the EET receptor antagonists and a selective EET synthesis inhibitor.

The results shown in Fig. 4 demonstrate the cardioprotective effect of POC and the effect of EET antagonists on the beneficial effect of POC. The chosen POC protocol produced a marked reduction in IS/AAR (10.1 ± 1.8%) similar to that produced by IPC and 14,15-EET. Similarly, pretreatment with both EET antagonists completely abolished the effect of POC when administered 5 min before the initial 30-s reperfusion period following the prolonged 60-min ischemic period (14,15-EEZE = 18.6 ± 1.0%, 14,15-EEZE-PEG = 22.0 ± 2.0%). Overall, these results clearly suggest a role for the endogenous EETs in both IPC and POC in the canine heart.

Effect of the 2 EET antagonists on the effect of POC in the canine heart. The EEZE compounds were given at 0.128 mg/kg 5 min before reperfusion just prior to POC, and both completely blocked the cardioprotective effect of POC to reduce IS/AAR. The occlusion/reperfusion intervals used to produce POC were 30 s three times given immediately after index ischemia.

DISCUSSION

Recent studies published by our laboratory (7) have shown that a putative selective EET receptor antagonist (4, 5), 14,15-EEZE, blocked the cardioprotective effect of 11,12-EET and 14,15-EET to reduce IS in the anesthetized dog heart when administered before the EETs at a dose that was without effect on IS when given alone. In a similar vein, 14,15-EEZE and 14,15-EEZE-PEG have been shown in the present study to block the cardioprotective effects of both IPC and POC, two powerful endogenous protective mechanisms in the canine heart. These data are the first to suggest that EETs are involved in triggering or mediating these two phenomena. These results are somewhat surprising since we previously reported (12) that administration of the EET synthesis inhibitor MS-PPOH (17) did not significantly inhibit the effects of IPC when administered before the IPC stimulus in dogs. It would stand to reason that an EET synthesis inhibitor would have an effect similar to that of a receptor blocker in these two models (12). It is possible that MS-PPOH is not selective for the CYP epoxygenase isoforms in the canine heart like it is in other tissues and species where it has been best characterized, such as the mouse and rat (6, 10). MS-PPOH may also block the CYP ω-hydroxylase in dogs as well as the CYP epoxygenases, which would render the CYP system ineffective in an IPC or POC heart where there are redundant mediators that could trigger the IPC or POC phenotype. Another possibility is that the dose of MS-PPOH used in our studies was not high enough to block the CYP epoxygenases in the canine heart. The latter hypothesis seems to be borne out in an additional group of dogs in which we found if we doubled the dose of MS-PPOH we were able to abrogate the potent cardioprotective effect of IPC (Table 3). Additional studies are needed to determine whether POC is also sensitive to a higher dose of MS-PPOH.

Current dogma suggests that the end effector(s) responsible for the cardioprotective effect of IPC and POC may be the result of opening the mitochondrial KATP channel (11, 13) and the subsequent inhibition of MPTP opening during the initial minutes of reperfusion (1, 9). Similarly, we and others have shown that opening the mitochondrial or sarcolemmal KATP channel mediates the protection produced by the EETs in dogs (12), rats (6), and mouse hearts (15, 16). Thus it is possible that these EET antagonists might be blocking the opening of the myocardial KATP channels directly and do not block the putative EET receptors. The EEZE compounds may have a more general effect to block the effects of numerous mediators that act via the KATP channel to produce cardioprotection. One series of experiments published in a recent paper from our laboratory (7) suggests that this may not be the case, however. We previously demonstrated (7) that 14,15-EEZE did not block the cardioprotective effect of diazoxide, a putative mitochondrial KATP channel opener in dogs, which suggests that the EETs are most likely acting more selectively on a receptor than on a more specific site such as the mitochondrial KATP channel. Perhaps the EEZE compounds have a selective effect on the sarcolemmal KATP channel. Obviously, more studies are needed to address these inconsistencies.

In conclusion, two selective EET antagonists, 14,15-EEZE and 14,15-EEZE-PEG, and an EET synthesis inhibitor, MS-PPOH, all blocked the cardioprotective effects of IPC and POC in the canine heart at doses that had no effect on IS when given alone. It is tempting to attribute this effect to the ability of the two EEZE compounds to block an EET receptor since these compounds do not inhibit EET synthesis (4) or block the cardioprotective effects of the KATP channel agonist diazoxide (7). Further studies are needed to determine the exact site and mechanism by which the EETs produce benefit to the heart and the mechanism by which the EEZE compounds block their protective effects.

Limitations of the study.

The major limitation of the study is the lack of EET measurements in the cardiac tissue or bloodstream draining the ischemic area. Although it is possible to measure EETs, particularly if their breakdown is inhibited such as when soluble epoxide hydrolase (sHE) inhibitors such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) are given (7), it becomes very difficult in a protocol such as the present one where there are no steady-state times when coronary blood flow is unchanging because of the numerous occlusion/reperfusion cycles needed to produce IPC and particularly POC.

In addition, because of the hydrophobic nature and possible enzymatic conjugation of the EETs at the cell membrane, it is unlikely that the measured EET concentrations are the absolute concentrations or local endogenous concentrations. The measured EET concentrations only represent EETs in free acid form in plasma, which are likely to be much lower than the absolute concentrations of endogenous EETs. Furthermore, these concentrations are also likely to be lower than exogenously added EETs in this study. Again, adding EETs exogenously may not result in effects from the total added concentrations, probably because of the loss of EETs to hydrophobic adsorption to the containers, association with other hydrophobic molecules, and/or how efficiently EETs get to their actual site of action.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant HL-74314-05 (G. J. Gross). Partial support was provided by NIH Grants GM-31278 (J. R. Falck) and HL-51055 (W. B. Campbell) and the Robert A. Welch Foundation (J. R. Falck).